Methods for detecting asymmetric dimethylarginine in a biological sample

ABSTRACT

The present invention provides methods of detecting asymmetric dimethylarginine (ADMA) in a sample, particularly a sample that may contain symmetrical dimethylarginine (SDMA) and/or arginine. The methods generally involve modifying any SDMA and arginine in the sample such that SDMA and arginine are readily distinguishable from ADMA; and detecting ADMA. The invention further provides antibodies specific for ADMA; antibodies specific for modified SDMA; and antibodies specific for modified arginine. The invention further provides kits for practicing the subject methods.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 60/426,677 filed Nov. 15, 2002, which application isincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The U.S. government may have certain rights in this invention, pursuantto grant no. R01 HL-63685 awarded by the National Institutes of Health,the National Heart, Lung and Blood Institute.

FIELD OF THE INVENTION

The present invention is in the field of assay methods, and inparticular assay methods for asymmetric dimethylarginine.

BACKGROUND OF THE INVENTION

Elevated asymmetric dimethylarginine (ADMA) levels have been observed invarious conditions, including hypertension, dyslipidemia, hyperglycemia,hyperhomocysteinemia, and renal failure, and are believed to be onecause of endothelial dysfunction in these conditions. Elevated plasmaADMA concentrations are also associated with an increased risk ofcardiovascular disease. As an endogenous inhibitor of nitric oxidesynthase, ADMA reduces nitric oxide (NO) production. NO plays a vitalpart in the vascular homeostasis. Aside from being the most potentvasodilator, NO inhibits platelet aggregation, smooth muscleproliferation, and adhesion molecule expression, which all play a partin atherogenesis. Throughout the last few years, basic scientificinvestigation has revealed the mechanism whereby ADMA becomes elevatedin patients with hypertension, hyperhomocysteinemia, hyperglycemia,hypercholesterolemia, and tobacco exposure.

Nevertheless, the field of ADMA is progressing slowly, mostly because ofthe laborious procedures required to quantify the molecule. A conclusivedemonstration of ADMA's clinical relevance requires clinical studieswith a great patient population. High pressure liquid chromatography(HPLC) is the most commonly used method to quantify ADMA. However, theuse of HPLC to quantitate ADMA suffers from several drawbacks. The mostcritical among them are efficiency and sensitivity. The labor-intensiveextraction and derivatization steps necessary for HPLC detection notonly makes the procedure more vulnerable to human errors, but also makesit unfitting for studies with a large sample size. Moreover, because thedetection limit for UV detectors seldom goes below the sub-micromolarlevel, intracellular ADMA level in disease states remains largelyunexplored, despite the fact that ADMA is generated intracellularly.

There is a need in the art for methods of detecting and quantitatingADMA that are simple, efficient, and readily adapted to high-throughputanalysis. The present invention addresses this need.

Literature

Takahashi (1968) J. Biol. Chem. 243:6171-6179; Stühlinger et al. (2002)J. Am. Med. Assoc. 287:1420-1426; U.S. Pat. No. 6,358,699; Teerlink etal. (2002) Anal. Biochem. 303:131-137; Dobashi et al. (2002) Analyst127:54-59; Vishwanathan et al. (2000) J. Chromatogr. B. Biomed. Sci.Appl. 748:157-166; Pi et al. (2000) J. Chromatogr. B. Biomed. Sci. Appl.742:199-203; Chen et al. (1997) J. Chromatogr. B. Biomed. Sci. Appl.692:467-471; Pettersson et al. (1997) J. Chromatogr. B. Biomed. Sci.Appl. 692:257-262.

SUMMARY OF THE INVENTION

The present invention provides methods of detecting asymmetricdimethylarginine (ADMA) in a sample, particularly a sample that maycontain symmetrical dimethylarginine (SDMA) and/or arginine. The methodsgenerally involve modifying any SDMA and arginine in the sample suchthat SDMA and arginine are readily distinguishable from ADMA; anddetecting ADMA. The invention further provides antibodies specific forADMA; antibodies specific for modified SDMA; and antibodies specific formodified arginine. The invention further provides kits for practicingthe subject methods.

FEATURES OF THE INVENTION

The present invention features a method of detecting asymmetricdimethylarginine (ADMA) in a sample comprising ADMA, symmetricdimethylarginine (SDMA), and arginine. The method generally involves: a)contacting a sample with an α-dicarbonyl compound, wherein said sampleis suspected of containing ADMA and at least one of SDMA and arginine,where the contacting step results in modification of the guanidinonitrogens of SDMA and the guanidino nitrogens of arginine, to producemodified SDMA and modified arginine; and b) detecting ADMA in thesample. In some embodiments, the α-dicarbonyl compound is phenylglyoxal.

In some embodiments, the method further involves the step of modifyingthe α-amino group of SDMA, ADMA, and arginine before the step ofmodifying the guanidino nitrogens of SDMA and the guanidino nitrogens ofarginine. In some of these embodiments, the α-amino group is modifiedwith a dye that provides a detectable signal.

In some embodiments, the detection step involves contacting the samplewith an antibody that binds specifically to dimethylarginines, whereinthe antibody does not bind to the modified SDMA. In other embodiments,the detection step involves contacting the sample with an antibody thatbinds specifically to ADMA. In some of these embodiments, the antibodyis detectably labeled.

In some embodiments, detection of ADMA is by high performance liquidchromatography. In other embodiments, detection of ADMA is by capillaryelectrophoresis.

The present invention further features an antibody that bindsspecifically to asymmetric dimethylarginine. In some embodiments, theantibody is detectably labeled.

The present invention further features an antibody that bindsspecifically to modified symmetric dimethylarginine (SDMA), wherein theguanidino nitrogens of the SDMA are modified by reaction with anα-dicarbonyl compound.

The present invention further features a kit for detecting asymmetricdimethylarginine (ADMA) in a sample. In some embodiments, the kitincludes an α-dicarbonyl agent that modifies the guanidino nitrogens ofSDMA and the guanidino nitrogens of arginine; and an antibody that bindsto ADMA. In other embodiments, the kit further includes an antibody thatbinds α-dicarbonyl-modified SDMA, and an antibody that bindsα-dicarbonyl-modified L-arginine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the structure of phenylglyoxal.

FIG. 2 depicts the reaction of phenylglyoxal with an arginine residue.

FIG. 3 depicts fluoro-nitro-benzoxadiazole labeled ADMA.

FIG. 4 depicts the reaction of SDMA with phenylglyoxal.

DEFINITIONS

Assay methods of the invention may be qualitative or quantitative. Thus,as used herein, the term “detection” refers to both qualitative andquantitative determinations, and therefore includes “measuring” and“determining a level of.”

A “biological sample” encompasses a variety of sample types obtainedfrom an individual and can be used in a diagnostic or monitoring assay.The definition encompasses blood, blood-derived samples, and otherliquid samples of biological origin, solid tissue samples such as abiopsy specimen or tissue cultures or cells derived therefrom and theprogeny thereof. The definition also includes samples that have beenmanipulated in any way after their procurement, such as by treatmentwith reagents, solubilization, or enrichment for certain components. Theterm “biological sample” encompasses a clinical sample, and alsoincludes cells in culture, cell supernatants, cell lysates, serum,plasma, cerebrospinal fluid, urine, saliva, biological fluid, and tissuesamples.

The term “binds specifically,” in the context of antibody binding,refers to high avidity and/or high affinity binding of an antibody to aspecific molecule, e.g., asymmetric dimethylarginine (ADMA), modifiedsymmetric dimethylarginine (SDMA), or modified arginine. For example,binding of an ADMA-specific antibody to ADMA is stronger than binding ofthe same antibody to arginine, SDMA, or modified SDMA, so that byadjusting binding conditions, the antibody binds almost exclusively toADMA and not to arginine, SDMA, modified arginine, or modified SDMA.Likewise, binding of an antibody specific to modified SDMA is strongerthan binding of the same antibody to arginine, ADMA, modified ADMA, orSDMA, so that by adjusting binding conditions, the antibody binds almostexclusively to SDMA and not to arginine, ADMA, modified ADMA, or SDMA.

Antibodies which bind specifically to ADMA, to modified SDMA, or tomodified arginine may be capable of binding other molecules at a weak,yet detectable, level (e.g., 10% or less of the binding shown to ADMA,modified SDMA, or modified arginine). Such weak binding, or backgroundbinding, is readily discernible from the specific antibody binding toADMA, modified SDMA, or modified arginine, e.g. by use of appropriatecontrols. In general, antibodies of the invention which bind to ADMA,modified SDMA, or modified arginine, with a binding affinity of 10⁻⁷mole/l or more, e.g., 10⁻⁸ mole/liters or more (e.g., 10⁻⁹ M, 10⁻¹⁰,10⁻¹¹, etc.) are said to bind specifically to ADMA, modified SDMA, ormodified arginine, respectively. In general, an antibody with a bindingaffinity of 10⁻⁶ mole/liters or less is not useful in that it will notbind an antigen at a detectable level using conventional methodologycurrently used.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anα-dicarbonyl compound” includes a plurality of such compounds andreference to “the modified SDMA” includes reference to one or moremodified SDMA molecules and equivalents thereof known to those skilledin the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of detecting asymmetricdimethylarginine (ADMA) in a sample, particularly in a sample that maycontain SDMA and/or arginine. The methods generally involve modifyingsymmetrical dimethylarginine (SDMA) and arginine such that SDMA andarginine are readily distinguishable from ADMA; and detecting ADMA. Theinvention further provides antibodies specific for ADMA, antibodiesspecific for modified SDMA, and antibodies specific for modifiedarginine. The invention further provides kits for practicing the subjectmethods.

Methods of Detecting ADMA in a Biological Sample

The present invention provides methods of detecting ADMA in a biologicalsample, particularly in a sample that may contain SDMA and/or arginine.The biological sample may comprise ADMA, SDMA, and arginine. The methodsinvolve modifying SDMA and arginine in such a way that the modified SDMAand modified arginine are readily distinguishable from ADMA. The methodsinvolve contacting a biological sample with an α-dicarbonyl compound,generating modified SDMA and modified arginine; and detecting ADMA inthe sample.

The structures of arginine, ADMA, and SDMA are shown below. The instantmethods involve modifying the guanidino nitrogen groups of SDMA and ofarginine with an α-dicarbonyl compound. SDMA and arginine are modifiedwith the α-dicarbonyl compound, while ADMA is not.

Acting as nucleophiles, the guanidino nitrogens of arginine attack thecarbonyl carbons of the α-dicarbonyl compound, generating a modifiedarginine that contains two modified nitrogen groups per guanidino group.Since the guanidino nitrogens on SDMA each take up one methyl group,they both still possess a hydrogen that is free to participate in anucleophilic reaction. Thus, as with arginine, the guanidino nitrogensof SDMA are also modified by the α-dicarbonyl compound to form a stableproduct. Without wishing to be bound by theory, it is believed thatparticipation of both guanidino nitrogens with the α-dicarbonyl compoundis crucial for modification of the compound because the resultant5-membered ring structure stabilizes the intermediate product. BecauseADMA has both methyl groups occupying the same guanidino nitrogen, theguanidino nitrogen is not available for reacting with the α-dicarbonylcompound, and ADMA does not react with an α-dicarbonyl compound to forma stable product.

Modifying SDMA and Arginine

Any of a variety of α-dicarbonyl compounds that are known in the art canbe used in the instant methods to modify guanidino nitrogens of SDMA andarginine. The structure of a generic α-dicarbonyl compound is shownbelow.

Suitable α-dicarbonyl compounds include, but are not limited to,dialdehydes, ketoaldehydes, and diketones. Non-limiting examples ofα-dicarbonyl compounds are biacetyl, pyruvic acid, glyoxal,methyglyoxal, deoxyosones, 3-deoxyosones, malondialdehyde,2-oxopropanal, phenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione.

In many embodiments, R is a bulky group, including, but not limited to,a cyclopentyl group, a substituted cyclopentyl group; a six-memberedring, such as phenyl, a substituted phenyl (e.g.,p-hydroxyphenylglyoxal, nitrophenylglyoxal, etc.), and the like. Inembodiments of particular interest, the α-dicarbonyl compound isphenylglyoxal. The structure of phenylglyoxal is shown below.

As one non-limiting example, where the α-dicarbonyl compound isphenylglyoxal, the reaction with arginine proceeds as follows:

The reaction with SDMA proceeds in a similar way.

In addition to reacting with the guanidino amine of arginine,phenylglyoxal has been reported to react with the α-amino group of thepeptides to give α-keto acyl peptides. Takahashi (1968) J. Biol. Chem.243:6171-6179. In the context of free amino acid, this observationindicates that phenylglyoxal will react with all α-amino groups of allamino acid. In some embodiments, the α-amino group is protected with aconventional labeling dye such as fluoro-nitro-benzoxadiazole (NBD-F),as described further below. Protection of the α-amino group ensures thatphenylglyoxal only reacts with the guanidino nitrogen. Many labeling dyemolecules are conjugated to the amino acid or proteins by reacting withthe α-amino group.

An α-dicarbonyl compound is contacted with a biological sample.Generally, the α-dicarbonyl compound is prepared in water, and the pH ofthe solution is adjusted to 9.0 with 1M NaOH. The α-dicarbonyl compoundis generally in a 10× stock solution in a concentration of from about 1mM to about 500 mM, e.g., from about 1 mM to about 10 mM, from about 10mM to about 50 mM, from about 50 mM to about 100 mM, from about 100 mMto about 200 mM, from about 200 mM to about 300 mM, from about 300 mM toabout 400 mM, or from about 400 mM to about 500 mM. In some embodiments,the α-dicarbonyl compound is in a 10× stock solution in a concentrationof from about 50 mM to about 100 mM. A solution containing theα-dicarbonyl compound is added to the biological sample in such a waythat the stock solution is diluted 10-fold.

The biological sample is contacted with the α-dicarbonyl compound, andthe reaction is allowed to proceed for a period of time of from about 15seconds to 2 hours, e.g., from about 15 seconds to about 60 seconds,from about 1 minute to about 15 minutes, from about 15 minutes to about30 minutes, from about 30 minutes to about 60 minutes, or from about 1hour to about 2 hours. In a particular embodiment, the reaction isallowed to proceed for 1 hour to about 2 hours in the dark at roomtemperature (e.g., at about 22° C.).

The reaction of components in the sample with the α-dicarbonyl compoundresults in modification of the guanidino nitrogen groups ofsubstantially all molecules of SDMA and arginine in the sample. Thus, atleast 80%, at least 90%, at least 95%, at least 98%, or at least 99% ofthe SDMA and arginine molecules in the sample are modified.

A variety of other reagents may be included in the assay. These includereagents such as buffers, salts, neutral proteins, e.g. albumin,detergents, etc. Reagents that improve the efficiency of the assay, suchas protease inhibitors, nuclease inhibitors, anti-microbial agents, etc.may also be used.

The instant assay methods may be designed a number of different ways.For example, the assay components of the method may be combined atsubstantially the same time or at different times. Incubations areperformed at any suitable temperature, typically between 4° and 40° C.Incubation periods are selected for optimum activity, but may also beoptimized to facilitate rapid high-throughput screening. In general, allcomponents are in solution.

An example of a biological sample is serum. However, any biologicalsample can be used. As one non-limiting example, serum or plasma isobtained from a blood sample of a patient, and from about 0.05 mL toabout 2.0 mL of serum or plasma is reacted with an α-dicarbonyl compoundsuch that the final concentration of the α-dicarbonyl compound in thesample is in a range of from about 30 mM to about 70 mM. The reaction isallowed to proceed for a suitable time, after which ADMA is detected.

Protecting the α-Amino Group

The α-dicarbonyl compound may react with the α-amino group of arginine,SDMA, and ADMA. In such cases, an optional step of derivatizing theα-amino group of arginine, SDMA, and ADMA is performed before themodification of the guanidino nitrogen groups of arginine and SDMA. Manymethods are known in the art for modifying (protecting) the α-aminogroup.

Protecting groups for α-amino groups are well known in the art include,but are not limited to, methyl, formyl, ethyl, acetyl, t-butyl, anisyl,benzyl, trifluoroacetyl, N-hydroxysuccinimide, t-Butyloxycarbonyl,benzoyl, 4-methylbenzyl, thioanizyl, thiocresyl, benzyloxymethyl,4-nitrophenyl, benzyloxycarbonyl, 2-nitrobenzoyl,2-nitrophenylsulphenyl, 4-toluenesulfphonyl, pentafluorophenyl,diphenylmethyl, 2-chlorobenzyloxycarbonyl, 2,4,5-trichlorophenyl,2-bromobenzyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, triphenylmethyl,2,2,5,7,8-pentamethyl-chroman-6-sulphonyl, 2-(4-nitrophenylsulfonyl)ethoxy carbonyl, 9-(2-sulfo)fluorenylmethyl carbamate,9-(2,7-dibromo)fluorenylmethyl carbamate,17-tetrabenzol[a,c,g,i]fluorenyl methyl carbamate,1,1-dioxobenzol[b]thiophen-2-ylmethyl carbamate, and the like.

Also suitable for use are groups that both protect the α-amino group andprovide for detection, e.g., the protecting group is a detectable label.Suitable fluorophores for this invention include fluorescein and itsanalogs, rhodamine and its analogs, cyanine and related polymethines andtheir analogs, and the like. Specific fluorophores which are suitablefor use with the present invention are Fluorescein isothiocyanate(FITC); 4-fluoro-7-nitrobenzofurazan (NBD-F); Texas Red™ (MolecularProbes, Inc.; Eugene, Oreg.); tetramethyl rhodamine isothiocyanate(TRITC); and Cyanine dyes, e.g., Cy3, Cy5, Cy5.5 Cy7, Cy7.5, Cy8 and Cy9(Biological Detection Systems, Pittsburgh, Pa.); phenyl-thiohydantoin(PTH), and phenylisothiocyanate (PITC). As one non-limiting example (seeabove), the α-amino group is reacted with4-fluoro-7-nitro-benzoxadiazole (NBD-F).

Detecting ADMA

ADMA is detected using any of a variety of methods. Such methodsinclude, but are not limited to, conversion of ADMA to citrulline,followed by spectrophotometric determination of citrulline;determination of ADMA with an antibody that binds dimethylarginines;determination of ADMA with an antibody specific for ADMA, using animmunological assay; high performance liquid chromatography; capillaryelectrophoresis; and the like. Detection of ADMA can be qualitative orquantitative. These methods are also useful for detecting derivatizedSDMA and L-arginine, which, after reacting as described above, are moreeasily distinguished from ADMA. Various methods are outlined below.

HPLC Methods

A variety of methods for detecting ADMA using HPLC are known in the art,any of which can be used in conjunction with an instant assay method.See, e.g., Teerlink et al. (2002) Anal. Biochem. 303:131-137; Dobashi etal. (2002) Analyst 127:54-59; Pi et al. (2000) J. Chromatogr. B. Biomed.Sci. Appl. 742:199-203; Chen et al. (1997) J. Chromatogr. B. Biomed.Sci. Appl. 692:467-471; Anderstam et al. (1997) J. Am. Soc. Nephrol.8:1487-1442; and Pettersson et al. (1997) J. Chromatogr. B. Biomed. Sci.Appl. 692:257-262.

Where HPLC is used to detect ADMA in a sample, the α-amino group of ADMAis modified (derivatized) with a detectable group, e.g., a fluorescentgroup. After the α-amino group is derivatized, the sample is treatedwith an α-dicarbonyl compound as described above. This will cause SDMAand arginine, but not ADMA, to be modified, so as to enhance theirseparation from ADMA when the sample is applied to an HPLC column.Separation of analytes is performed on the HPLC using a solvent. Theanalytes, including ADMA, are eluted, and the amount of ADMA isdetermined by measuring the amount of the detectable label in eachanalyte peak. The amount of ADMA can be determined by determining thepeak area.

Solid phase extraction (SPE) of the biological sample is frequentlycarried out to clean up the sample prior to applying the sample to theHPLC column. A variety of SPE columns are available and can be used inconjunction with the instant methods. Suitable SPE columns include anOasis MCX SPE column (Waters); a Bond Elute SCX 50 mg column (VarianInc., Palo Alto, Calif.); and the like. These columns retainpositively-charged compounds, which are then collected by eluting thecolumn with a weak base such as trimethylamine.

The following is one non-limiting example of a method of detecting ADMAusing HPLC. In this method, the biological sample is cleaned up on anSPE column; the primary amine containing compounds (eg. ADMA, SDMA,arginine) are derivatized with an ortho-phthaldialdehyde reagentcontaining 3-mercaptopropionic acid; and, after this derivatization, thesample is treated with an α-dicarbonyl compound and then the derivatizedsample is applied to a reversed-phase HPLC, and the peak correspondingto ADMA is detected with fluorescence detection. The peak correspondingto ADMA is clearly distinguished from peaks corresponding to L-arginineor SDMA.

In this example, the biological sample is first cleaned up on a SPEcolumn. 0.2 ml of biological sample (e.g., plasma, serum, urine,cerebrospinal fluid, and the like) is mixed with 0.1 ml of an internalstandard and 0.7 ml phosphate buffered saline. The sample is applied toan Oasis MCX SPE column (Waters). After application of the sample, thecolumn is washed consecutively with 1.0 ml of 100 mM HCl and 1.0 mlmethanol. Analytes are eluted from the column in 3.0-ml tubes with 1.0ml concentrated ammonia (or triethylamine)/water/methanol (10/40/50).The solvent is removed from the analytes by evaporation with nitrogen ata temperature of 60-80° C. The residue is dissolved in 0.1 ml water toform the analyte sample.

In this example, analytes eluted from the SPE column are derivatizedwith ortho-phthaldialdehyde (OPA). To the 0.1 ml analyte sample is added0.1 ml OPA working solution. OPA stock solution is prepared bydissolving 10 mg OPA in 0.2 ml methanol, followed by addition of 1.8 mlof a 200 mM potassium borate buffer (pH 9.5) and 10 μl3-mercaptopropionic acid. The OPA working solution is prepared byfive-fold dilution of the stock solution with borate buffer. The finalconcentrations of OPA and 3-mercaptopropionic acid in the working OPAsolution are 7.5 mM and 11.5 mM, respectively. After the OPA workingsolution is added to the analyte sample, the samples are derivatizedwith an α-dicarbonyl compound, and are applied to an HPLC column.

In this example, HPLC is performed on a Symmetry C18 column (3.9×150 mm;5 μm particle size; 100 Å pore size) with a 10×3 mm guard column packedwith the same stationary phase. Mobile phase A consists of 50 mMpotassium phosphate buffer (pH 6.5), containing 8.7% acetonitrile, andmobile phase B is acetonitrile/water (50/50, v/v). Separation isperformed under isocratic conditions with 100% mobile phase A at a flowrate of 1.1 muminute and a column temperature of 30° C. After elution ofthe last analyte, closely related compounds are eluted with a strongsolvent flush (50% B from 20 to 22 minutes). Fluorescence is measured atexcitation and emission wavelengths of 340 an 455 nm, respectively.Peaks are quantified on the basis of peak area.

Those skilled in the art will recognize that many modifications to theabove example of an HPLC method are possible.

Capillary Electrophoresis

Where CE is used to detect ADMA content of a sample, α-amino groups ofall of the amino acids (including that of ADMA) are modified with alabeling group (e.g., NBD-F). Subsequently, SDMA and L-arginine arederivatized with an α-dicarbonyl compound.

Methods of using capillary electrophoresis to detect ADMA are known inthe art, and any known method can be used in conjunction with theinstant methods. See, e.g., Vallance et al. (1992) Lancet 339:572-575;and Causse et al. (2000) J. Chromatogr. B. Biomed. Sci. Appl. 741:77-83.

Immunoassays

A variety of immunoassays can be designed that make use of the abilityto distinguish modified SDMA and modified arginine from ADMA. Incarrying out an immunoassay, one or more of the following antibodieswill be used: (1) antibodies that bind dimethylarginines, i.e., thatspecifically bind both SDMA and ADMA and that do not discriminatebetween SDMA and ADMA; (2) antibodies that specifically bind ADMA; (3)antibodies that specifically bind modified SDMA; (4) antibodies thatbind both modified SDMA and modified L-arginine; and (5) antibodies thatspecifically bind modified arginine.

Modification of arginine and SDMA as described above modifies thesemolecules such that they no longer react with antibodies that bind bothSDMA and ADMA (i.e., antibodies specific for dimethylarginines). Thus,in some embodiments, determination of ADMA following modification ofarginine and SDMA is carried out with conventional immunological assays,using antibodies specific for dimethylarginines. Because substantiallyall of the SDMA in the sample is modified by the α-dicarbonyl compoundduring the modification reaction, and because the modified SDMA is notrecognized by antibodies specific for dimethylarginines, such antibodieswill only detect ADMA in the sample.

Detection of ADMA can also be carried out using an antibody specific forADMA (e.g., antibody that binds ADMA, but that does not substantiallybind to SDMA, arginine, or modified SDMA), as described in more detailbelow.

Immunoassays can also be carried out using antibodies specific formodified SDMA and antibodies specific for modified arginine, asdescribed in more detail below.

Detection with a specific antibody is carried out using well-knownmethods. In general, the antibody is detectably labeled, either directlyor indirectly. Direct labels include radioisotopes (e.g., ¹²⁵I; ³⁵S, andthe like); enzymes whose products are detectable (e.g., luciferase,β-galactosidase, horse radish peroxidase, and the like); fluorescentlabels (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, andthe like); fluorescence emitting metals, e.g., ¹⁵²Eu, or others of thelanthanide series, attached to the antibody through metal chelatinggroups such as EDTA; chemiluminescent compounds, e.g., luminol,isoluminol, acridinium salts, and the like; bioluminescent compounds,e.g., luciferin; fluorescent proteins; and the like. Fluorescentproteins include, but are not limited to, a green fluorescent protein(GFP), including, but not limited to, a “humanized” version of a GFP,e.g., wherein codons of the naturally-occurring nucleotide sequence arechanged to more closely match human codon bias; a GFP derived fromAequoria victoria or a derivative thereof, e.g., a “humanized”derivative such as Enhanced GFP, which are available commercially, e.g.,from Clontech, Inc.; a GFP from another species such as Renillareniformis, Renilla mulleri, or Ptilosarcus guernyi, as described in,e.g., WO 99/49019 and Peelle et al. (2001) J. Protein Chem. 20:507-519;“humanized” recombinant GFP (hrGFP) (Stratagene); any of a variety offluorescent and colored proteins from Anthozoan species, as describedin, e.g., Matz et al. (1999) Nature Biotechnol. 17:969-973; and thelike.

Indirect labels include second antibodies that bind to antibodiesspecific for ADMA or dimethylarginines, wherein the second antibody islabeled as described above. Indirect labels also include members ofspecific binding pairs, e.g., biotin-avidin, and the like, as are wellknown in the art. For example, the primary antibody may be conjugated tobiotin, with horseradish peroxidase-conjugated avidin added as a secondstage reagent. Final detection uses a substrate that undergoes a colorchange in the presence of the peroxidase. Alternatively, the secondaryantibody conjugated to a fluorescent compound, e.g. fluorescein,rhodamine, Texas red, etc. The absence or presence of antibody bindingmay be determined by various methods, including spectrophotometricdetection, fluorimetry, radiography, scintillation counting, etc.

Quantification can be carried out using any known method, including, butnot limited to, enzyme-linked immunosorbent assay (ELISA);radioimmunoassay (RIA); and the like. In general, quantitation isaccomplished by comparing the level of ADMA detected in the sample withthe amount of ADMA present in a standard curve.

In many embodiments, an assay will employ a specific antibody (e.g., anantibody specific for ADMA, or an antibody specific fordimethylarginines), which antibody is bound to a solid support, such asa test strip. Test strips are in provided in a variety of shapes (e.g.,rectangles, squares, circles, etc.) and materials (e.g. nylon, polyvinylpyrollidone, polyester, polycarbonate, cellulose acetate,polyethersulfone, nitrocellulose, and the like). Such assays can bedesigned in any of a number of ways. In general, a specific antibody isbound to a test strip, and the antibody bound to the test strip capturesADMA in the sample. For example, a sample is applied to one end of atest strip, and the components of the sample are allowed to migrate bycapillary action or lateral flow. Methods and devices for lateral flowseparation, detection, and quantitation are known in the art. See, e.g.,U.S. Pat. Nos. 5,569,608; 6,297,020; and 6,403,383. Once the ADMA iscaptured by the bound antibody, a second antibody that is detectablylabeled is used to detect the captured ADMA.

In another embodiment, a sample that has been modified as describedabove is spotted onto a membrane (e.g., nylon, polyvinyl pyrollidone,polyester, polycarbonate, cellulose acetate, polyethersulfone,nitrocellulose, and the like). Typically, several spots that correspondto increasing dilutions of the sample are applied. For example, serial1:2 dilutions are made and spotted onto the membrane. Detectably labeledantibody specific for ADMA or detectably labeled antibody specific fordimethylarginines is used to quantitate the level of ADMA in the sample.

In other embodiments, an assay will employ an antibody specific formodified SDMA, which antibody is bound to a solid support. For example,a test strip is used that includes, in order from a first end to asecond end, a sample loading region, a first antibody region thatincludes an antibody specific for modified SDMA, and a second regionthat includes an antibody that binds ADMA (either an antibody specificfor ADMA or an antibody specific for dimethylarginines). A sample isapplied to the sample loading region, and the components of the sampleare allowed to migrate toward the second end of the test strip bycapillary action or lateral flow. Modified SDMA is captured in the firstregion, leaving ADMA free to migrate to the second region, where it iscaptured. The captured ADMA is detected as described above.

In other embodiments, an assay will employ an antibody specific formodified SDMA and an antibody specific for modified arginine, whichantibodies are bound to a solid support. For example, a test strip isused that includes, in order from a first end to a second end, a sampleloading region, a first antibody region that includes an antibodyspecific for modified SDMA, a second region that includes an antibodythat binds modified arginine, and a third region that includes anantibody that binds ADMA (either an antibody specific for ADMA or anantibody specific for dimethylarginines). A sample is applied to thesample loading region, and the components of the sample are allowed tomigrate toward the second end of the test strip by capillary action orlateral flow. Modified SDMA is captured in the first region, modifiedarginine is captured in the second region, leaving ADMA free to migrateto the third region, where it is captured. The captured ADMA is detectedas described above.

As an alternative, the antibody specific for modified SDMA and modifiedarginine can be combined into a first region. For example, a test stripis used that includes, in order from a first end to a second end, asample loading region, a first antibody region that includes an antibodyspecific for modified SDMA and an antibody that binds modified arginine;and a second region that includes an antibody that binds ADMA (either anantibody specific for ADMA or an antibody specific fordimethylarginines). A sample is applied to the sample loading region,and the components of the sample are allowed to migrate toward thesecond end of the test strip by capillary action or lateral flow.Modified SDMA and modified arginine are captured in the first region,leaving ADMA free to migrate to the second region, where it is captured.The captured ADMA is detected as described above.

Other Assays

Other methods of detecting ADMA include liquid chromatography-tandemmass spectrometry. See, e.g., Vishwanathan et al. (2000) J. Chromatogr.B. Biomed. Sci. Appl. 748:157-166.

Assays for detecting ADMA can also be carried out on a sample that isdepleted of modified SDMA and modified arginine. In these embodiments,antibodies specific for modified SDMA and antibodies specific formodified arginine are used to remove modified SDMA and modified argininefrom a sample that has been reacted with an α-dicarbonyl compound, asdescribed above. Once modified SDMA and modified arginine are removedfrom the sample, leaving ADMA, the ADMA is detected using any knownmethod, including the above-mentioned methods. For example, antibodiesspecific for modified SDMA and antibodies specific for modified arginineare coupled to an insoluble support (e.g., immobilized), and, afterreacting the biological sample with the α-dicarbonyl compound, themodified sample is contacted with the immobilized antibodies. After asuitable time, the modified sample is separated from the immobilizedantibodies, and ADMA is detected in the sample. Antibodies can beimmobilized on any of a variety of insoluble supports, including, butnot limited to, beads, including magnetic beads, polystyrene beads; anaffinity column matrix; a membrane; a plastic surface; and the like.

Antibodies

The present invention further provides antibodies that specifically bindADMA. Antibodies that specifically bind ADMA do not detectably bindarginine, SDMA, modified arginine, or modified SDMA, or bind only at abackground level. Antibodies specific for ADMA are useful for detectingADMA in a sample that may comprise ADMA, SDMA, and arginine.

Alternatively, antibodies specific for modified SDMA and modifiedarginine are used together with antibodies directed againstdimethylarginines (after modification of SDMA in the sample, onlyunmodified ADMA would be detected by these antibodies). Accordingly, theα-dicarbonyl modification can be used to enhance the specificity of anyantibody binding to ADMA.

The present invention further provides antibodies that specifically bindmodified SDMA, where the guanidino nitrogen residues are modified byreaction with an α-dicarbonyl compound, as described above. Antibodiesthat specifically bind modified SDMA do not detectably bind arginine,ADMA, or unmodified SDMA, or bind only at a background level.

The present invention further provides antibodies that specifically bindmodified arginine, where the guanidino nitrogen residues are modified byreaction with an α-dicarbonyl compound, as described above.

In many embodiments, a subject antibody is isolated, e.g., is in anenvironment other than its naturally-occurring environment. Suitableantibodies are obtained by immunizing a host animal with ADMA, withmodified SDMA, or with modified arginine, as appropriate. Suitable hostanimals include mouse, rat sheep, goat, hamster, rabbit, etc.

For preparation of polyclonal antibodies, the first step is immunizationof the host animal with the antigen, i.e., ADMA, modified SDMA, ormodified arginine, where the antigen will preferably be in substantiallypure form, comprising less than about 1% contaminant. To increase theimmune response of the host animal, the antigen is typically coupled toa carrier, and may be combined with an adjuvant, where suitableadjuvants include alum, dextran, sulfate, large polymeric anions, oil &water emulsions, e.g. Freund's adjuvant, Freund's complete adjuvant, andthe like. The antigen is typically conjugated to a carrier molecule,e.g., a synthetic carrier molecule protein, a synthetic antigen, keyholelimpet hemocyanin, and the like. A variety of hosts may be immunized toproduce the polyclonal antibodies. Such hosts include rabbits, guineapigs, rodents, e.g. mice, rats, sheep, goats, and the like. The antigenis administered to the host, e.g., intradermally, or intraperitoneally,with an initial dosage followed by one or more, usually at least two,additional booster dosages. Following immunization, the blood from thehost will be collected, followed by separation of the serum from theblood cells. The Ig present in the resultant antiserum may be furtherfractionated using known methods, such as ammonium salt fractionation,DEAE chromatography, and the like.

Monoclonal antibodies are produced by conventional techniques.Generally, the spleen and/or lymph nodes of an immunized host animalprovide a source of plasma cells. The plasma cells are immortalized byfusion with myeloma cells to produce hybridoma cells. Culturesupernatant from individual hybridomas is screened using standardtechniques to identify those producing antibodies with the desiredspecificity. The antibody may be purified from the hybridoma cellsupernatants or ascites fluid by conventional techniques, e.g. affinitychromatography using protein bound to an insoluble support, protein Asepharose, etc.

The antibody may be produced as a single chain, instead of the normalmultimeric structure. Single chain antibodies are described in Jost etal. (1994) J.B.C. 269:26267-73, and others. DNA sequences encoding thevariable region of the heavy chain and the variable region of the lightchain are ligated to a spacer encoding at least about 4 amino acids ofsmall neutral amino acids, including glycine and/or serine. The proteinencoded by this fusion allows assembly of a functional variable regionthat retains the specificity and affinity of the original antibody.

Also provided are “artificial” antibodies, e.g., antibodies and antibodyfragments produced and selected in vitro. In some embodiments, suchantibodies are displayed on the surface of a bacteriophage or otherviral particle. In many embodiments, such artificial antibodies arepresent as fusion proteins with a viral or bacteriophage structuralprotein, including, but not limited to, M13 gene III protein. Methods ofproducing such artificial antibodies are well known in the art. See,e.g., U.S. Pat. Nos. 5,516,637; 5,223,409; 5,658,727; 5,667,988;5,498,538; 5,403,484; 5,571,698; and 5,625,033.

Also of interest in certain embodiments are humanized antibodies.Methods of humanizing antibodies are known in the art. The humanizedantibody may be the product of an animal having transgenic humanimmunoglobulin constant region genes (see for example InternationalPatent Applications WO 90/10077 and WO 90/04036). Alternatively, theantibody of interest may be engineered by recombinant DNA techniques tosubstitute the CH1, CH2, CH3, hinge domains, and/or the framework domainwith the corresponding human sequence (see WO 92/02190).

The use of Ig cDNA for construction of chimeric immunoglobulin genes isknown in the art (Liu et al. (1987) Proc. Natl. Acad. Sci. USA. 84:3439and (1987) J. Immunol. 139:3521). mRNA is isolated from a hybridoma orother cell producing the antibody and used to produce cDNA. The cDNA ofinterest may be amplified by the polymerase chain reaction usingspecific primers (U.S. Pat. Nos. 4,683,195 and 4,683,202).Alternatively, a library is made and screened to isolate the sequence ofinterest. The DNA sequence encoding the variable region of the antibodyis then fused to human constant region sequences. The sequences of humanconstant regions genes may be found in Kabat et al. (1991) Sequences ofProteins of Immunological Interest, N.I.H. publication no. 91-3242.Human C region genes are readily available from known clones. The choiceof isotype will be guided by the desired effector functions, such ascomplement fixation, or activity in antibody-dependent cellularcytotoxicity. Preferred isotypes are IgG1, IgG3 and IgG4. Either of thehuman light chain constant regions, kappa or lambda, may be used. Thechimeric, humanized antibody is then expressed by conventional methods.Other methods for preparing chimeric antibodies are described in, e.g.,U.S. Pat. No. 5,565,332.

Antibody fragments, such as Fv, F(ab′)₂ and Fab may be prepared bycleavage of the intact protein, e.g. by protease or chemical cleavage.Alternatively, a truncated gene is designed. For example, a chimericgene encoding a portion of the F(ab′)₂ fragment would include DNAsequences encoding the CH1 domain and hinge region of the H chain,followed by a translational stop codon to yield the truncated molecule.

Consensus sequences of H and L J regions may be used to designoligonucleotides for use as primers to introduce useful restrictionsites into the J region for subsequent linkage of V region segments tohuman C region segments. C region cDNA can be modified by site directedmutagenesis to place a restriction site at the analogous position in thehuman sequence.

Expression vectors include plasmids, retroviruses, YACs, EBV derivedepisomes, and the like. A convenient vector is one that encodes afunctionally complete human CH or CL immunoglobulin sequence, withappropriate restriction sites engineered so that any VH or VL sequencecan be easily inserted and expressed. In such vectors, splicing usuallyoccurs between the splice donor site in the inserted J region and thesplice acceptor site preceding the human C region, and also at thesplice regions that occur within the human CH exons. Polyadenylation andtranscription termination occur at native chromosomal sites downstreamof the coding regions. The resulting chimeric antibody may be joined toany strong promoter, including retroviral LTRs, e.g. SV-40 earlypromoter, (Okayama et al. (1983) Mol. Cell. Bio. 3:280), Rous sarcomavirus LTR (Gorman et al. (1982) P.N.A.S. 79:6777), and moloney murineleukemia virus LTR (Grosschedl et al. (1985) Cell 41:885); native Igpromoters, etc.

In some embodiments, a subject antibody is detectably labeled. Adetectable label is any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Detectable labels include radioisotopes; enzymes whoseproducts are detectable (e.g., luciferase, β-galactosidase, and thelike); fluorescent labels (e.g., fluorescein isothiocyanate, rhodamine,phycoerythrin, and the like); fluorescence emitting metals, e.g., ¹⁵²Eu,or others of the lanthanide series, attached to the antibody throughmetal chelating groups such as EDTA; chemiluminescent compounds, e.g.,luminol, isoluminol, acridinium salts, and the like; bioluminescentcompounds, e.g., luciferin, aequorin (a green fluorescent protein); amagnetic bead; colloidal gold or colored glass or plastic beads (e.g.,polystyrene, polypropylene, latex, etc.) or other labels that can bedetected by mass spectroscopy, NMR spectroscopy, or other analyticalmeans known in the art.

A subject antibody can be labeled with a fluorescent protein asdescribed in Matz et al., Nature Biotechnology (October 1999)17:969-973; a green fluorescent protein (GFP), including a “humanized”GFP; a GFP from Aequoria victoria or fluorescent mutant thereof, e.g.,as described in U.S. Pat. No. 6,066,476; 6,020,192; 5,985,577;5,976,796; 5,968,750; 5,968,738; 5,958,713; 5,919,445; 5,874,304, thedisclosures of which are herein incorporated by reference; a GFP fromanother species such as Renilla reniformis, Renilla mulleri, orPtilosarcus guernyi, as described in, e.g., WO 99/49019 and Peelle etal. (2001) J. Protein Chem. 20:507-519; “humanized” recombinant GFP(hrGFP) (Stratagene); other fluorescent dyes, e.g., coumarin and itsderivatives, e.g. 7-amino-4-methylcoumarin, aminocoumarin, bodipy dyes,such as Bodipy FL, cascade blue, fluorescein and its derivatives, e.g.fluorescein isothiocyanate, Oregon green, rhodamine dyes, e.g. texasred, tetramethylrhodamine, eosins and erythrosins, cyanine dyes, e.g.Cy3 and Cy5, macrocyclic chelates of lanthanide ions, e.g. quantum dye,etc., chemilumescent dyes, e.g., luciferases.

In some embodiments, a subject antibody is labeled with an indirectlydetectable label. An indirectly detectable label includes a member of aspecific binding pair. Specific binding pairs include, but are notlimited to, biotin-avidin, biotin-streptavidin, digoxin and antidigoxinand the like.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple calorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, e.g., in various dipstick assays, conjugated gold oftenappears pink, while various conjugated beads appear the color of thebead.

In some embodiments, a subject antibody is coupled (directly or througha linker) to an insoluble support. The antibody may be attached(coupled) to an insoluble support, including, but not limited to, aplastic surface (e.g., a polystyrene plate); a membrane (e.g.,nitrocellulose, nylon, polyvinyl pyrollidone, polyester, polycarbonate,cellulose acetate, polyethersulfone, etc); a bead (e.g., a magneticbead, a plastic bead); a colloidal particle; an affinity column matrix;and the like.

Detection using a subject antibody involves use of direct labels orindirect labels. Indirect labels include second antibodies specific fora subject antibody (e.g., specific for a heavy chain constant region ofa subject antibody), wherein the second antibody is labeled as describedabove; and members of specific binding pairs, e.g., biotin-avidin, andthe like. The biological sample may be brought into contact with animmobilized on a solid support or carrier, such as a membrane, beads,and the like, that is capable of immobilizing cells, cell particles, orsoluble proteins. The support may then be washed with suitable buffers,followed by contacting with a detectably-labeled subject antibody.Detection methods are known in the art and will be chosen as appropriateto the signal emitted by the detectable label. Detection is generallyaccomplished in comparison to suitable controls, and to appropriatestandards.

Kits

The present invention further provides kits for practicing the subjectmethods. A subject kit will include an α-dicarbonyl compound formodifying the guanidino nitrogen groups of SDMA and arginine; and anantibody. Suitable antibodies include those that bind specifically toADMA; antibodies that specifically bind dimethylarginines (e.g.,antibodies that bind both SDMA and ADMA); antibodies that specificallybind modified SDMA; antibodies that bind α-dicarbonyl forms of SDMA andα-dicarbonyl forms of L-arginine; and antibodies that specifically bindmodified arginine. The subject kit components are typically present in asuitable storage medium, e.g., buffered solution, typically in asuitable container. A subject kit may further include membranes forcarrying out an immunological assay, e.g., a test strip.

In addition to the above components, the subject kits will furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, compact disk (CD), digital versatile disk, etc., onwhich the information has been recorded. Yet another means that may bepresent is a website address which may be used via the internet toaccess the information at a removed site. Any convenient means may bepresent in the kits.

Utility

The subject methods and kits are useful for detecting ADMA in abiological sample. The subject methods are useful for determining alevel of ADMA in a biological sample, and therefore are useful indiagnostic methods for various disorders, including methods fordetermining the risk of developing a disorder.

The subject methods are useful for diagnosing various disorders forwhich elevated ADMA levels are diagnostic, including, but not limitedto, hypertension, hyperhomocysteinemia, hyperglycemia,hypercholesterolemia, insulin resistance, renal insufficiency,congestive heart failure, atherosclerosis, transplant arteriopathy, andendothelial dysfunction and the like. For example, an increase in thelevel of ADMA, compared to the level in a normal, healthy individual,indicates that the individual is at risk for vascular dysfunction ordisease.

The subject methods are also useful for determining the extent, theseverity, the progression, or stage, of a disorder for which an elevatedADMA level is diagnostic. A biological sample is taken at a single timepoint and the level of ADMA is compared to a chart of standard normalvalues for ADMA. The severity of the disorder is assessed by comparingthe detected levels of ADMA in the biological sample with levels of ADMAin a standard curve, and associating the level with the severity of thedisorder. The severity of the disorder may allow the selection of moreefficacious therapies, for example a mild elevation of ADMA in ahypercholesterolemic subject may indicate that lifestyle changes aresufficient therapy, whereas a severe elevation of ADMA would indicatethat drug therapy should be employed.

The subject methods are useful for monitoring progression of a disorderfor which an elevated ADMA level is diagnostic. Determining ADMA levelsat different times is used to monitor the progression of the disorder. Abiological sample is taken from the individual and tested at a frequencyof once per week, twice weekly, once per month, bimonthly, once everythree months, once every four months, once every 6 months, or once ayear, depending on various factors. In these embodiments, the level ofADMA in a test sample is compared to the level of ADMA in a previoussample(s). An increase in the level of ADMA in a test sample, comparedto one or more previous test samples, indicates that the disease isincreasing in severity. The rate of increase in the level of ADMA is anindication of the rate of progression of the disease. A reduction inADMA may be seen with treatment (e.g., insulin-resistant subjects havingelevated ADMA levels exhibit reduced ADMA levels following treatmentwith metformin).

The subject methods are also useful for determining the risk that anindividual will develop a disorder for which an elevated ADMA level isdiagnostic. An elevated ADMA level, compared to a control value for anormal healthy individual, may indicate that the individual is at riskfor developing a disorder for which elevated ADMA levels are diagnostic.ADMA has been shown to be predictive of cardiovascular mortality inpatients with coronary artery disease or renal insufficiency (Zoccali C,Bode-Boger S, Mallamaci F, Benedetto F, Tripepi G, Malatino L,Cataliotti A, Bellanuova I, Fermo I, Frolich J, Boger R. Plasmaconcentration of asymmetrical dimethylarginine and mortality in patientswith end-stage renal disease: a prospective study. Lancet. 2001 Dec.22-29;358(9299):2113-7. Valkonen V P, Paiva H, Salonen J T, Lakka T A,Lehtimaki T, Laakso J, Laaksonen R. Risk of acute coronary events andserum concentration of asymmetrical dimethylarginine. Lancet. 2001 Dec.22-29;358(9299):2127-8.) Additional tests may be recommended todetermine whether an individual is developing a given disorder. In viewof the test results, an appropriate treatment regimen may berecommended.

The subject methods are also useful for determining the response of anindividual to treatment for a disorder for which an elevated ADMA levelis diagnostic. Measurements of ADMA levels are used to determine whethera patient is responding to treatment. ADMA levels are measured beforeand after a treatment to determine if the treatment is efficacious. ADMAlevels are also determined during the course of the treatment, todetermine whether the treatment slows the progression of the disease,and to what extent the treatment slows the progression of the disease.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric.

Example 1 Method for Modifying SDMA and Arginine

There are two isoforms of dimethylarginine—asymmetric (SDMA) andsymmetric (ADMA), depending on how the methyl groups are distributed onthe guanidino function group of arginine. Any detection method for ADMAneeds to be able to distinguish among ADMA, SDMA, and arginine, whichare structurally very similar. Some HPLC system can achieve theresolution; however, it suffers from the drawbacks mentioned above.Available antibodies against dimethylarginine, though improvingefficiency and sensitivity, indiscriminately bind to both ADMA and SDMA(e.g., ab413, Abcam, Cambridge Science Park, UK). The following approachutilizes a chemical reaction that specifically modifies SDMA, but notADMA. SDMA, but not ADMA, will react with α-dicarbonyl compounds,leaving the modified SDMA to sufficiently differ from ADMA such that anantibody directed against dimethylarginines can then be used toselectively detect ADMA. This method also enhances the selectivity ofantibodies or engineered molecules that preferentially (but notselectively) detect ADMA.

A large number of α-dicarbonyl compounds (dialdehydes, ketoaldehydes anddiketones) have been used in the past four decades to modify arginineresidue in proteins. Before site-specific mutation became available,such modification indicates whether an arginine residue is part of anenzyme's active site. Phenylglyoxal (FIG. 1) is an α-dicarbonyl compoundstill in use today for active site determination. Under mild conditions,phenylglyoxal reacts with the arginine residue. Acting as nucleophiles,the guanidino nitrogens from arginine attack the carbonyl carbons,forming a five-member ring structure. The unstable dialcoholintermediate then reacts with another phenylglyoxal, giving rise to aproduct that contains two phenylglyoxal moieties per guanidino group(FIG. 2).

This product is relatively stable at acidic pH's, but at pH>10, thereaction was observed to be reversible. Typically, phenylglyoxal isdissolved in water and the pH is adjusted to 9.0 with 1M NaOH. Asolution containing phenylglyoxal is then added to the sample in such away that the stock solution is diluted 10-fold. The reaction proceeds inthe dark at room temperature for 60-180 minutes (Reference: Tawfik D S,Walter J M, Modification of arginine side chains withp-hydroxyphenylglyoxal, The Proteins Protocol Handbook 2002, 2^(nd)edition, Humana Press Inc.)

In addition to reacting with the guanidino amine of arginine,phenylglyoxal has been reported to react with the α-amino group of thepeptides to give α-keto acyl peptides. In the context of free aminoacid, this observation indicates that phenylglyoxal will react with allα-amino group of all amino acid. To ensure that phenylglyoxal onlyreacts with the guanidino nitrogen, we can protect the α-amino groupwith a conventional labeling dye such as fuoro-nitro-benzoxadiazole(NBD-F). Many labeling dye molecules are conjugated to the amino acid orproteins by binding to the α-amino group.

After the α-amino group of SDMA and ADMA are protected by reacting withNBD-F, phenylglyoxal would be added to the mixture. Since the guanidinonitrogens on SDMA each take up one methyl group, they both still possessa hydrogen free to participate in nucleophilic reaction. ADMA, on theother hand, has both methyl groups occupying the same guanidinonitrogen, disabling that nitrogen from further reaction (FIG. 3).

Participation of both guanidino nitrogens in the reaction withphenylglyoxal is crucial for the ring formation, and hence stability, ofthe final product. Thus, SDMA, but not ADMA, would react withphenylglyoxal, via the reaction outlined in FIG. 4.

According to the above scheme, phenylglyoxal would react with both SDMAand arginine. An antibody against dimethylarginines could be used tospecifically detect ADMA, by first reacting the samples withphenylglyoxal, which modifies SDMA, but not ADMA.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. A method of detecting asymmetric dimethylarginine (ADMA) in a samplecomprising ADMA and at least one of symmetric dimethylarginine (SDMA)and arginine, the method comprising the steps of: a) contacting thesample with an agent that protects the α-amino group of ADMA and theα-amino group of the at least one of SDMA and arginine, generating asample comprising ADMA having a protected α-amino group and at least oneof SDMA and arginine having a protected α-amino group; b) contacting thesample generated in step (a) with an α-dicarbonyl compound, wherein saidα-dicarbonyl compound modifies the guanidino nitrogens of SDMA and theguanidino nitrogens of arginine, producing modified SDMA and modifiedarginine, wherein said modified SDMA and said modified arginine aredistinguishable from ADMA; and c) detecting ADMA in the sample generatedin step (b).
 2. The method of claim 1, wherein said α-dicarbonylcompound is phenylglyoxal.
 3. The method of claim 1, wherein the agentthat protects the α-amino group is a dye that provides a detectablesignal.
 4. The method of claim 3, wherein the dye is a fluorophore. 5.The method of claim 4, wherein the fluorophore isfluoro-7-nitrobenzofurazan.
 6. The method of claim 1, wherein saiddetecting step comprises contacting the sample with an antibody thatbinds specifically to ADMA, to SDMA, or to both ADMA and SDMA, whereinsaid antibody does not bind to the modified SDMA.
 7. The method of claim6, wherein the antibody is detectably labeled.
 8. The method of claim 1,wherein said detecting step comprises contacting the sample with anantibody that binds specifically to the α-amino group-modified ADMA. 9.The method of claim 8, further comprising detecting one or more ofmodified SDMA and modified arginine, wherein said detection of one ormore of modified SDMA and modified arginine comprises contacting thesample with an antibody that binds specifically to one or more ofmodified SDMA and modified arginine.
 10. The method of claim 1, whereinsaid ADMA is detected by high performance liquid chromatography.
 11. Themethod of claim 1, wherein said ADMA is detected by capillaryelectrophoresis.
 12. The method of claim 1, wherein the α-dicarbonylcompound is selected from biacetyl, pyruvic acid, glyoxal, methyglyoxal,deoxyosones, 3-deoxyosones, malondialdehyde, 2-oxopropanal,phenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione.
 13. The methodof claim 1, wherein the sample is a biological sample.
 14. The method ofclaim 13, wherein the biological sample is serum or plasma.
 15. Themethod of claim 13, wherein the biological sample is subjected to solidphase extraction before step (a) to clean up the sample.
 16. The methodof claim 1, wherein the agent that protects the α-amino group isortho-phthaldialdehyde.